Development of pyrF-based gene knockout systems for genome-wide manipulation of the archaea Haloferax mediterranei and Haloarcula hispanica

Hailong Liu; Jing Han; Xiaoqing Liu; Jian Zhou; Hua Xiang

doi:10.1016/j.jgg.2011.05.003

Volume 38 Issue 6

Jun. 2011

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Article Navigation > Journal of Genetics and Genomics > 2011 > 38(6): 261-269

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Development of pyrF-based gene knockout systems for genome-wide manipulation of the archaea Haloferax mediterranei and Haloarcula hispanica

doi: 10.1016/j.jgg.2011.05.003

State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China

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Corresponding author: E-mail address: xiangh@sun.im.ac.cn (Hua Xiang)
Received Date: 2011-04-08
Accepted Date: 2011-05-03
Rev Recd Date: 2011-05-03

Available Online: 2011-05-17

Publish Date: 2011-06-20

Abstract

Abstract

The haloarchaea Haloferax mediterranei and Haloarcula hispanica are both polyhydroxyalkanoate producers in the domain Archaea, and they are becoming increasingly attractive for research and biotechnology due to their unique genetic and metabolic features. To accelerate their genome-level genetic and metabolic analyses, we have developed specific and highly efficient gene knockout systems for these two haloarchaea. These gene knockout systems consist of a suicide plasmid vector with the pyrF gene as the selection marker and a uracil auxotrophic haloarchaeon (▵pyrF) as the host. For in-frame deletion of a target gene, the suicide plasmid carrying the flanking region of the target gene was transferred into the corresponding ▵pyrF host. After positive selection of the single-crossover integration recombinants (pop-in) on AS-168SY medium without uracil and counterselection of the double-crossover pyrF-excised recombinants (pop-out) with 5-fluoroorotic acid (5-FOA), the target gene knockout mutants were confirmed by PCR and Southern blot analysis. We have demonstrated the effectiveness of these systems by knocking out thecrtB gene which encodes a phytoene synthase in these haloarchaea. In conclusion, these well-developed knockout systems would greatly accelerate the functional genomic research of these halophilic archaea.
- Knockout system,
- Pop-in/pop-out method,
- Haloarchaea

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References(27)

References

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